The Gram negative subgingival anaerobic bacterium Porphyromonas gingivalis is capable of avoiding host innate immune recognition, ensuring both its survival and that of bystander bacterial species in dental plaque. It has been associated with causing dysbiosis of the oral microflora in animal models of infection that results in periodontitis. Our goal was to identify novel P. gingivalis genetic determinants required for immune evasion. In preliminary data, we describe the construction of a transposon-based P. gingivalis mutant library, which was subjected to an in vitro screen we developed to isolate mutants that incur a strong pro-inflammatory response, distinct from non-inflammatory wild-type strains, including the parental strain employed in this study, 33277. We obtained a mutant, called J5-c5, that stimulated a pro-inflammatory response via activation of the immune receptor TLR4. Modulation of the host TLR4 response is a primary means by which P. gingivalis avoids immune elimination. P. gingivalis can modify its lipid A, the TLR4 ligand, from a penta-acylated TLR4-agonistic structure to tetra-acylated TLR4-anatagonistic and TLR4-inert structures. We hypothesized that lipid A in J5-c5 is largely penta-acylated, which was confirmed by MALDI-TOF chromatography. Lack of tetra-acylated lipid A is consistent with loss of a functional lipid A deacylase, a critical enzyme that remains unidentified. Whole genome sequencing revealed the presence and location of five transposon insertions in J5-c5. Our goal now has become to identify the specific insertion site responsible for loss of deacylase activity, and to elucidate the reason behind this loss. In Aim 1, we will construct five separate 33277 mutants, each with a tetQ insertion in the same location as one of the five transposon insertions. The mutant with the same phenotype as J5-c5 will reveal the locus required for deacylation. The disrupted locus could be required for structure, such as a gene coding for the deacylase itself or a protein required for deacylase assembly; or could play an essential role in regulation of deacylase gene transcription, all of these possibilities will be investigated in this proposal. In Aim 2, we will compare global RNA expression patterns between wild-type and J5-C5, using RNAseq, to determine whether dysregulation of deacylase gene expression explains loss of deacylase activity. Of note, two lipid A deacylases identified so far in other bacteria are transcriptionally regulated. Differential RNA expression will also identify the gene encoding the deacylase, or the gene required for deacylase assembly, that has been rendered non-functional in J5-c5 either directly by the insertion or indirectly by regulation. Deacylase gene identification will enable the study of its expression levels in P. gingivalis strains that exhibit different levels of virulence, and strains from different in vivo locations. It will be crucial to investigate whether and how the gene is regulated. Importantly, it will fill a critical gap in understanding the molecular basis of an essential mechanism used by P. gingivalis to evade the host immune response